Capillary electrophoresis (CE) is one of the most powerful separation methods. Analysis can be conducted even when samples are sub-nanoliter in volume (Ewing, A. G.; Wallingford, R. A.; Olefirowicz, T. M., Anal. Chem., 61, 292A-303A, 1989).
In capillary electrophoresis, a capillary containing buffer is suspended between two reservoirs filled with buffer. An electric field is applied across the two ends of the capillary. Analytes in a sample introduced at the high potential end migrate toward the low potential end under the influence of the electric field. Typically, capillary electrophoresis is carried out with a 0-60 KV DC power supply. When an analyte is about to exit the capillary, it can be detected by various techniques.
Commonly used methods for detecting analytes in CE include absorption, fluorescence, electrochemical, and mass spectrometric detection (Ewing et al. supra). Among these methods, UV-vis absorption detectors are the most popular because of their versatility and simplicity, and because they are usually supplied with commercial CE systems.
However, on-column absorption schemes in CE can only detect about 10.sup.-5 M to 10.sup.-6 M of an analyte in a sample due to the limited pathlength, low intensity, and the presence of stray light when using an incoherent light source (Bruin, G. J. M.; Stegeman, G.; Van Asten, A. C.; Xu, X.; Kxaak, J. C.; Poppe, H. J., Chromatogr., 559, 163-181, 1991).
One way to improve the detection limit in CE is to increase the effective pathlength. Taylor and Yeung increased the effective pathlength of their absorbance detectors by directing the light beam along the capillary axis to obtain an approximately 60-fold improvement in the pathlength (Taylor, J. A.; Yeung, E. S., J. Chromatogr., 550, 831-837, 1991). The reduced zone lengths associated with CE unit axial beam detection and the choice of electrolytic buffers is limited to those with a refractive index higher than that of the column walls.
Liquid chromatography (LC) is also a powerful analytical technique. LC separates analytes in a mixture based on the repetitive distribution of the molecules of the analytes between a mobile and a stationary phase. The mobile phase is a liquid through which the analytes pass. In high-performance liquid chromatography (HPLC), the driving force for the movement of liquid and analytes is primarily the pressure difference between the two ends of the chromatographic column. Mho and Yeung disclosed a detection method for ion chromatography based on double-beam Laser-Excited Indirect Fluorometry (Mho, S.; Yeung, E. S.; Anal. Chem., 57(12), 2253-2256, 1985). Detection methods based on absorption for analytes in LC are similar to those used in CE. As with any absorption measurement, noise reduction will lead to increased sensitivity.
Lasers are not widely used in conjunction with CE or LC because of their instability which increases noise, thereby hindering the detection of low analyte concentrations in small samples. One way of reducing noise is to use a dual beam detector system. In a conventional double-beam absorption detector, the light output is split into a sample and a reference beam. The resulting photocurrents, or voltages, are either subtracted from each other or divided. Subtraction requires extremely fine adjustment of the two beams to equal intensities and requires identical detector and amplifier characteristics for good noise suppression. An analog divider is typically used for conventional division and suffers from the poor performance.
Hobbs, et al. describe a double-beam laser absorption system based on all-electronic noise suppression (Hobbs, P. C. D., SPIE Proc., Roy, R., Ed., 1376, 216-221, 1991; Haller, K. L. and Hobbs, P. C. D., SPIE Proc., Feary, B. L., Ed., 1435, 298-309, 1991; Hobbs, P. C. D., Optics & Photonics News, 17-23, April 1991). The system functions by subtracting the signal from the reference photocurrents under feedback control to cancel spurious modulation of the laser beam and excess noise, which is the noise above the shot-noise level. Multiple spectral scans of a sample in a cell containing I.sub.2 were subjected to analog low frequency (100 Hz high pass) filtering and signal-averaging (1000 individual sweeps) to achieve a noise-equivalent absorption (i.e., noise in absorption) of 2.times.10.sup.-7 (Haller, K. L. and Hobbs, P. C. D., SPIE Proc., Feary, B. L., Ed., 1435, 298-309, 1991).
Hobbs (U.S. Pat. No. 5,134,276) discloses electrical circuits for noise suppression in such a system. The systems described by Hobbs et al. in the aforementioned documents were, however, applied in spectroscopy and are not related to CE or LC, which are low frequency operations.
To achieve noise reduction, Hobbs et al. scanned the output repetitively and averaged the signals. While that process may improve the output accuracy, it prevents the system from providing "real-time" or "on-column" data of the changing levels of analyte in the sample. Furthermore, in technologies such as biotechnology, analyses often have to be conducted on samples which are small in volume and contain very dilute analytes. By requiring multiple scans, such systems may not be able to accurately analyze small, dilute samples.
As a result, there is a need for a capillary separation system which is capable of providing highly sensitive, real-time detection of analytes which may be dilute and/or provided in small samples.